P1377: TRANSCRIPTOME-WIDE ANALYSIS OF T CELLS DURING EX VIVO EXPANSION AS PER CURRENT CAR T CELL MANUFACTURING PROTOCOL REVEALS SPECIFIC CHANGES IN T CELL SIGNALING AND HOMING
Authors: Harish Sudarsanam, Maximilian Radtke, Raymund Buhmann, Vladan Vučinić, Rami Abou-Jamra, Reinhard Henschler
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Ex vivo expansion is a necessary process in the current CAR T cell manufacturing protocols. Different ex vivo expansion conditions and duration of expansion are under investigation to enhance functionality and longer persistence of CAR T cell products upon infusion. Recent studies have revealed that shorter culture periods lead to a higher percentage of less differentiated stem-like memory T cells (Tscm) and central memory T cells (Tcm) in the final product. Multiple clinical trials with short and rapid manufacturing protocols (with 3 days of manufacturing) for CAR T cells are underway. However, the changes induced in the T cells during ex vivo manufacturing conditions and their impact on the functionality of CAR T cells upon infusion are not deeply understood. Aims: We aimed to characterize transcriptome-wide changes in ex vivo expanded T cells compared to freshly isolated peripheral blood T cells, in order to gain insight into dynamics and functional changes induced in CAR T cells after transfer of cells into culture environment. Methods: Peripheral blood T cells were isolated from healthy male donors aged 20-29 between, using CD3 Fab-Traceless Affinity Cell Selection (TACS) Agarose Columns. Cells were cultured for 7 days in X-VIVO 15, 5% hAB Serum, 50U IL-2 and were activated using 10µl/ml of anti-CD3/CD28 for the initial 3 days. Total RNA was isolated from fresh T cells and from T cells cultured for 4, 12 and 24 hours, 3 and 7 days. Total RNA sequencing was performed on Illumina NovaSeq and differential gene expression as well as functional analysis was performed to identify major cellular and molecular changes. Results: Dynamic hierarchical clustering identified 20 gene clusters with different transcriptional regulation patterns over time, including fast and persistent upregulation and downregulation, as well as several patterns of biphasic and triphasic modulation in gene expression levels. We next identified common functionalities at early and later time points and within these profiles. ERK1/2 cascade, MAPK cascade, STAT signaling, and cellular response to cytokines and chemokines were the top enriched signaling pathways at the 4 hrs time point, indicating rapid transcriptional response in T cells to ex vivo expansion conditions. Focused analysis revealed a sudden (4 hrs) and persistent early response in key genes responsible for leukocyte migration and adhesion, including rapid and persistent > 3-fold downregulation of integrin α4 and integrin β1, and a reversible 6-fold downregulation of integrins β2 and β7. Expression of chemokine receptors CXCR4 and CCR6 was persistently downregulated approx. 10 and 6-fold, respectively from 4 hrs on until end of culture, whereas CCR7 remained at its initial level and CXCR3 was steadily increasing about 3-fold. In contrast, other migration- and cytoskeleton-regulating genes including Rho family GTPases did not show significant differential expression. At later time points (>24 hrs), the highly regulated transcripts were genes acting on cell cycle and cell division. Several transcription factors which are associated with regulating these gene sets, as well as micro-RNA and other non-coding RNAs were also differentially regulated. Summary/Conclusion: This study provides evidence of major changes in T cell gene expression, at very early phases and also at later stages of ex vivo expansion. This gives a basis for understanding deficits in adhesion, migration and homing of cultured T cells and provides insights into the impact of ex vivo expansion on the functionality and long-term persistence of CAR T cells upon infusion into patients.Keywords: Ex vivo expansion, T cell, Cellular therapy, CAR-T